This invention relates to methods and compositions for detecting the existence or measuring the concentration of bacterial contamination in food products.
Ground beef and chicken are susceptible to rapid spoilage by psychotropic bacteria which thrive at refrigeration temperatures. As a result, these products have very short shelf-lives which are directly related to the initial concentration of contaminating bacteria.
Current methods for measuring the concentrations of bacterial contamination in ground beef and chicken include the standard plate count (Difco Laboratories) as well as the Petri Film system (3M) (see generally, Compendium of Methods for the Microbiological Examination of Foods, Third Edition, Edited by Carl Vanderzant and Don F. Splittstoesser, Compiled by the APHA Technical Committee on Microbiological Methods for Foods). These methods require around 48 hours of incubation in a 35xc2x0 C. incubator before the results can be read. Both methods utilize a solid nutrient base to support the growth of individual cells into bacterial colonies. Many food-borne bacteria are incapable of growing into colonies on these surfaces when incubated at 35xc2x0 C.; thus, the concentrations of total viable bacteria measured by the above methods may be underestimated.
In addition, the long incubation periods of these methods can cause these food products to remain in storage for several days until the concentrations of contaminating bacteria are known. If these tests could be completed in a shorter period of time it would allow companies to release their products sooner so as to lower costs, increase sales, and provide better product to the consumer.
There have been attempts to measure the bacterial concentration in food by measuring specific metabolic by-products of individual microorganisms. These methods include: electrical impedance assays, ATP assays, antibody-based assays, and carbon-14 labelled substrate assays. Indicators of microbial growth have also been used to monitor the growth of target microbes which change color only after growth of the target microbe is detected. These indicators normally react chemically with a metabolic by-product produced by the target microbes resulting in a color change in the medium. Examples of chemicals which change color in the presence of pH changes associated with growth include phenol red, bromocresol blue, and neutral red. For example, Golber, U.S. Pat. No. 3,206,317, uses phenol red, a chemical which changes color in the presence of acidic waste products produced by the target microbe. Berger et al., U.S. Pat. No. 3,496,066, describes the use of compounds which bacteria convert to dyestuffs, e.g., tropinones and dioxanes, Bochner, U.S. Pat. No. 4,129,483 describes using a non-biodegradable substance (tetrazolium) which is chemically reduced to produce a color change. In all of these examples, the indicator is a compound which does not serve as a source of a required nutrient.
Edberg (U.S. Pat. No. 4,925,789), incorporated by reference herein, describes a selective growth medium for a microbe containing a nutrient indicator which can only be metabolized by a target microbe. When metabolized by a target microbe, the nutrient indicator releases a moiety which imparts a detectable change to the medium.
The present invention relates to a bacterial growth medium and methods for detecting the existence or measuring the concentration of bacteria in a test sample. The claimed medium and methods measure viable bacteria as a function of the activities of several classes of bacterial enzymes, including, but not limited to, phosphatases, glycosidases (such as glucosidases), and aminopeptidases. The presence of at least one of these groups of enzymes in any given bacterial species will be detected by the appearance of a detectable signal such as a fluorescent signal. Therefore, this invention is useful in detecting the existence or measuring the concentration of total viable bacteria or at least a multitude of viable bacteria in a test sample in a single assay. In specific examples, cocktails of enzyme substrates are made to measure the concentration of bacterial contamination in food products, such as ground beef and chicken.
Thus, in one aspect, the invention features a bacterial growth medium containing three or more different enzyme substrates each hydrolysed by a different bacterial enzyme to cause or produce a detectable signal.
In a preferred embodiment, the three or more different enzyme substrates each has both a nutrient moiety and a detectable moiety linked together by a covalent bond. Each of these enzyme substrates is hydrolysed by a different bacterial enzyme to produce a separate detectable moiety which causes or produces a detectable signal in the medium. In a further preferred embodiment, the detectable signals caused or produced are of identical type.
By xe2x80x9cmediumxe2x80x9d is meant a solid, powder or liquid mixture which contains all or substantially all of the nutrients necessary to support bacterial growth. Amino acids, minerals, vitamins and other elements known to those skilled in the art to be necessary for bacterial growth are provided in the medium, including, but not limited to, those disclosed in U.S. application Ser. Nos. 08/334,788 (abandoned in favor of C-I-P application Ser. No. 08/423,134 filed Apr. 18, 1995 and issued U.S. Pat. No. 5,610,029 on Mar. 11, 1997) and 08/335,149, (issued as a U.S. Pat. No. 5,620,865 on Apr. 15, 1997) both filed on Nov. 4, 1994, incorporated by reference herein. In a preferred embodiment, the medium is liquid.
For example, the following components are provided in the medium in approximately the amounts indicated. Those in the art will understand that not every component is required. Components may also be substituted with other components of similar properties. The amounts of components may also be varied.
Amino acids may be provided from a variety of sources. These can be provided from natural sources (e.g., extracts of organisms), as mixtures, or in purified form. The natural mixtures may contain varying amounts of such amino acids and vitamins. Not all amino acids must be provided, and the relative amount of each can vary. For general guidance, specific amounts of such amino acids and vitamins are indicated below. These amounts are for guidance only and are not limiting in this invention. Those in the art will recognize that many different combinations of amino acids and vitamins can be used in the medium of this invention. The lists provided below exemplify just one such example. Normally, only amino acids which cannot be synthesized endogenously by the microorganisms to be detected must be provided. However, other amino acids may be provided without departing from the medium of the invention.
The medium preferably includes at least the following amino acids in approximately the following amounts (per liter of medium): Alanine (0.015 to 0.60 grams), Arginine (0.080 to 3.2 grams), Aspartic Acid (0.018 to 0.72 grams), Cystine (0.09 to 3.6 grams), Glutamic Acid (0.030 to 1.20 grams), Glycine (0.050 to 2.00 grams), Histidine (0.025 to 1.00 grams), Isoleucine (0.035 to 1.40 grams), Leucine (0.040 to 1.60 grams), Lysine (0.050 to 2.00 grams), Methionine (0.01 to 0.50 grams), Phenylalanine (0.01 to 0.90 grams), Proline (0.02 to 2.80 grams), Serine (0.01 to 0.40 grams), Threonine (0.01 to 1.10 grams), Tryptophan (0.002 to 0.26 grams), Tyrosine (0.01 to 1.20 grams), and Valine (0.02 to 1.10 grams).
Salts may be provided as a source of ions upon dissociation. Such salts may include (per liter of medium): potassium chloride (e.g., about 0.5 to 1.5 grams); copper sulfate (e.g., about 40 to 50 xcexcg); ammonium acetate or ammonium sulfate (e.g., about 4.0 to 6.0 grams); potassium iodide (e.g., about 50.0 to 150.0 xcexcg); ferric chloride (e.g., about 150.0 to 250.0 xcexcg); manganese sulfate (e.g., about 300.0 to 500.0 xcexcg); sodium molybdate (e.g., about 150.0 to 250.0 xcexcg); zinc sulfate (e.g. about 300.0 to 500.0 xcexcg); and sodium chloride (e.g. about 0.05 to 0.15 g).
Other inorganic moieties may be included to aid microbial growth. These include the following (to the extent not already provided in the above sources of various chemical entities and described in amounts per liter): Phosphorus (about 0.5 mg), Potassium (about 0.4 mg), Sodium (about 30 to 60 mg), and trace amounts of Calcium, Magnesium, Aluminum, Barium, Chloride, Cobalt, Copper, Iron, Lead, Manganese, Suffate, Sulfur, Tin and Zinc.
Vitamins required for growth and reproduction of the microorganism sought to be detected may also be provided. These can be provided in a pure form or as part of a more complex medium. Such vitamins may be present in approximately the following amounts (per liter of medium): Biotin (about 0.15 to 60 xcexcg), Pantothenic Acid (about 15.0 to 65.0 xcexcg), Pyridoxine (about 2.0 to 9.0 xcexcg), Riboflavin (about 10.0 to 50.0 xcexcg), Folic acid (about 5.00 to 50.00 xcexcg), Thiamine (about 10.0 to 50.0 xcexcg), Vitamin B12 (about 0.20 to 0.50 xcexcg), and Niacin (about 15.0 to 55.0 xcexcg).
By xe2x80x9cbacterial enzymexe2x80x9d is meant an enzyme whose enzymatic activity such as the ability to hydrolyse a substrate or a plurality of substrates is characteristic of a bacterium or a plurality of bacteria. In this invention, the enzymatic activities of a bacterial enzyme or bacterial enzymes are used to detect or measure the concentration of bacteria in a test sample. The bacterial enzymes include all those known to one skilled in the art, including, but not limited to, those listed in Enzymes, 3rd edition, edited by Malcolm Dixson, Edwin C. Webb, C. J. R. Thorne, and K. F. Tipton, 1979, Academic Press, U.S.A. In a preferred embodiment, the bacterial enzyme is selected from the group consisting of alkaline phosphatase, acid phosphatase, esterase, lipase, N-acetyl-xcex2-D-galactosaminidase, N-acetyl-xcex2-D-glucosaminidase, Neuraminidase, L-arabinopyranosidase, xcex2-D-fucosidase, xcex1-L-fucosidase, xcex2-L-fucosidase, xcex1-D-galactosidase, xcex2-D-galactosidase, xcex1-D-glucosidase, xcex2-D-glucosidase, xcex2-D-glucuronidase, xcex1-D-mannosidase, pyrophosphatase, sulfatase, xcex2-D-xylosidase, peptidase (preferably an aminopeptidase, more preferably an (L or D amino acid)xe2x80x94aminopeptidase), trypsin, chymotrypsin, and phosphohydrolase.
By xe2x80x9csubstratexe2x80x9d is meant a molecule or substance on which a bacterial enzyme acts. The enzymatic reaction usually involves hydrolysing one or more covalent bonds, forming one or more covalent bonds, or both. A covalent bond in the substrate between the nutrient moiety and the detectable moiety is hydrolysed by a bacterial enzyme to produce a separate detectable moiety. The substrates include all those known to one skilled in the art, including, but not limited to, those in the product listing of AerChem, Inc. with detectable moieties attached thereto (see Table I).
By xe2x80x9cnutrient moietyxe2x80x9d is meant a molecule or substance which is a nutrient or metabolic source for a bacterium, including, but not limited to, vitamins, minerals (e.g., phosphorus in the form of phosphate), trace elements, amino acids (e.g., L-alanine), carbon (e.g., glucose), or nitrogen.
By xe2x80x9cdetectable signalxe2x80x9d is meant a characteristic change in a medium or sample that is observable or measurable by physical, chemical, or biological means known to those skilled in the art. Such a detectable signal may be a change in emission or absorbance of visible or invisible light or radio waves at a certain wavelength, electrical conductivity, hybridization, enzymatic reaction, emission of gas, or odor. A detectable signal may also be a change in physical state such as between solid, liquid and gas. In preferred embodiments, detectable signals include a change in color or fluorescent emission of the medium.
By xe2x80x9cidentical type of detectable signalxe2x80x9d is meant that the separate detectable moieties hydrolysed from different enzyme substrates cause or produce detectable signals that are measurable by the same or substantially the same physical, chemical or biological parameter, including, but not limited to, color, fluorescent emission, odor, enzymatic reaction, hybridization, or electric conductivity (although the intensity or quantity of signals caused or produced by different separate detectable moieties may be different). For example, yellow colors of different intensity would be considered of the identical type. Color change and fluorescence would not be considered to be identical type of detectable signal.
By xe2x80x9cdetectable moietyxe2x80x9d is meant a molecule or substance which can be covalently linked to a nutrient moiety or exists as a separate entity by itself. The detectable moiety does not cause or produce a detectable signal when it is covalently bonded to a nutrient moiety. However, when an enzyme from a bacterium hydrolyses the substrate, a detectable moiety is released and causes or produces a detectable signal. In preferred embodiments, the detectable moieties are chromogens which produce a color change observable in the visible wavelength range or fluoresces when properly excited by an external energy source. Examples of detectable moieties include, but are not limited to, orthonitrophenyl, phenolphthalein, and 4-methylumbelliferone moieties.
The invention also features a method of using the medium to detect the existence or measure the concentration of bacterial contamination in a test sample. The medium is inoculated with the test sample and incubated under a condition suitable for bacterial growth for a certain time period (preferably no more than 24 hours, more preferably no more than 15 hrs, even more preferably no more than 10 hours). Then the detectable signal is measured as an indication of the concentration of bacteria in the test sample. Using this method, a detectable signal is produced when at least one of the three or more different bacterial enzymes is or are present in the bacteria which are incubating in the medium.
By xe2x80x9ctest samplexe2x80x9d is meant a piece, fraction, aliquot, droplet, portion, fragment, volume, or tidbit taken from a food product such as ground beef or chicken, a human or animal test subject, a soil, water, air or other environmental source, or any other source whose bacterial concentration is to be measured. A test sample may be taken from a source using techniques known to one skilled in the art, including, but not limited to, those described or referred to in Compendium of Methods for the Microbiological Examination of Foods, Third Edition, Edited by Carl Vanderzant and Don F. Splittstoesser, Compiled by the APHA Technical Committee on Microbiological Methods for Foods, incorporated by reference herein.
By xe2x80x9cbacteriaxe2x80x9d is meant one or more viable bacteria existing or co-existing collectively in a test sample. The term may refer to a single bacterium (e.g., Aeromonas hydrophilia, Aeromonas caviae, Aeromonas sobria, Streptococcus uberis, Enterococcus faecium, Enterococcus faecalis, Bacillus sphaericus, Pseudomonas fluorescens, Pseudomonas putida, Serratia liquefaciens, Lactococcus lactis, Xanthomonas maltophilia, Staphylococcus simulans, Staphylococcus hominis, Streptococcus constellatus, Streptococcus anginosus, Escherichia coli, Staphylococcus aureus, Mycobacterium fortuitum, and Klebsiella pneumonia), a genus of bacteria (e.g., streptococci, pseudomonas and enterococci), a number of related species of bacteria (e.g., coliforms), an even larger group of bacteria having a common characteristic (e.g., all gram-negative bacteria), a group of bacteria commonly found in a food product, an animal or human subject, or an environmental source, or a combination of two or more bacteria listed above. The bacteria include those described or referred to in Bergey""s Manual of Systematic Bacteriology, 1989, Williams and Wilkins, U.S.A., incorporated by reference herein.
In preferred embodiments, one of the substrates is hydrolysed by the enzyme alkaline phosphatase; another substrate is hydrolysed by the enzyme glycosidase, including, but not limited to, xcex2-D-glucosidase; and a third substrate is hydrolysed by a peptidase (preferably an aminopeptidase, more preferably an (L or D amino acid)xe2x80x94aminopeptidase), including, but not limited to, L-alanine aminopeptidase; the detectable moiety is a fluorescent moiety such that when the detectable moiety is hydrolysed from a substrate, it causes or produces a fluorescent signal; the medium contains at least the following three substrates: 4-methylumbelliferyl phosphate, 4-methylumbelliferyl-xcex2-D-glucoside and L-alanine-7-amido-4-methyl coumarin; and the medium is inoculated with a test sample from a food product, including, but not limited to, ground beef, chicken, milk, dairy products, and drinking water.
In another aspect, the invention features a bacterial growth medium containing two or more different enzyme substrates each hydrolysed by a different bacterial enzyme to cause or produce an identical type of detectable signal.
In a preferred embodiment, the two or more different substrates each has both a nutrient moiety and a detectable moiety linked together by a covalent bond. Each of these substrates is hydrolysed by a different bacterial enzyme to produce a separate detectable moiety which causes or produces an identical type of detectable signal.
The invention also features a method of using the medium to detect the existence or measure the concentration of bacteria in a test sample. The medium is inoculated with the test sample and incubated under a condition suitable for bacterial growth for a certain time period (preferably no more than 24 hours, more preferably no more than 15 hrs, even more preferably no more than 10 hours). Then the detectable signal is measured as an indication of the concentration of bacterial contamination in the test sample. Using this method, a detectable signal is produced when at least one of the two or more different bacterial enzymes is present in the incubation medium.
In preferred embodiments, the substrates are hydrolysed by an enzyme selected from the group consisting of alkaline phosphatase, glycosidase (which includes, but is not limited to, xcex2-D-glucosidase), and peptidase (preferably an aminopeptidase, more preferably an (L or D amino acid)xe2x80x94aminopeptidase, including, but not limited to, L-alanine aminopeptidase); and the detectable moiety and the medium are analogous to those noted above.
In other embodiments, the invention uses the apparatus described by Naqui et al. in U.S. patent application Ser. No. 08/201,110 (issues as U.S. Pat. No. 5,518,892 on May 21, 1996), incorporated by reference herein, to quantify the concentration of bacterial contamination. An example of such an apparatus is sold by Idexx Laboratories Inc. under the name of Quanti Tray(trademark). The quantifying step involves providing a test sample in a liquid form. The sample is placed or dispensed into the sample holding bag described by Naqui et al., and mixed with a medium to allow and promote growth of target bacteria within individual compartments. The mixture is incubated and the quantity and quality of the color or fluorescence change in each compartment is detected. The quantity and quality of positive compartment (i.e., a compartment having a detectable color or fluorescence change) is compared to a most probable number table which relates that value to the bacterial concentration of the test sample.
This invention has many advantages over the methods currently used to measure bacterial contamination. One advantage is its relatively short time to results. Certain psychotropic bacteria grow very slowly and can take from 48 to 72 hours before their colonies become large enough to count on an agar plate. However, countable colonies need not be present for the results of Applicant""s test to be read. The fluorescent color produced by these bacteria in the invention appears much faster than their corresponding colonies which results in a much shorter detection time. Applicant""s test can reduce the incubation period to 24 hours or less.
Another advantage of the invention has over standard methods is the absence of interference by bacterial overgrowth. This is a particular problem when Bacillus species are present because they tend to grow over other bacterial colonies in such a way that the plate is unreadable. The Bacillus species are common in food, particularly those that have been heat treated, such as pasteurized milk. This problem is avoided in the invention because it does not depend on counting individual bacterial colonies.
This invention can be used in microbiology laboratories involved in end product testing and/or quality control of food products, the meat and poultry industries, the dairy industry, and the water industry. The invention may be used to measure the concentration of total viable bacteria in drinking water.
This invention also relates to a growth medium and methods for detecting or measuring the concentration of yeasts, fungi, or other eukaryotic microorganisms in a test sample using a formulated medium and steps like those described above.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.